The invention relates to a method for homogeneous detection and analysis of nucleic acid sequences by use of single-labeled oligonucleotide probes whose fluorescence emission changes in response to probe-target hybridization and dissociation, and more particularly, to methods for analyzing one or multiple nucleic acid loci using said probes. This invention further relates to the use of fluorescence changes in single-labeled probes for melting curve analysis, genotyping, and pathogen detection, and to a method for quantification of specific sequences in real-time monitoring of nucleic acid amplification.
Probe hybridization is a widely used method for the detection, analysis, and quantification of nucleic acid sequences. Common techniques include Southern hybridization, dot blotting, gel-shift assays, and solution-based homogeneous assays, and are often coupled with polymerase chain reaction (PCR). The basic devices used in these techniques include electrophoresis gels, DNA arrays immobilized on surfaces of glass slides, beads, membranes or microtiter plates, and instrumentation for homogeneous assays such as the LightCycler system (Roche Molecular Biochemicals), the ABI PRISM7700 sequence detection system (PE Applied Biosystems), and the iCycler system (Bio-Rad Laboratories). Homogeneous assays, detection methods that are coupled with amplification processes, perform amplification and analysis in one continuous flow, eliminating or minimizing the need to transfer samples between the two processes. One key element that makes homogenous assays work is a reporter signal generated from probe-target hybridization that is detectable without the need to wash away free probe.
Current homogeneous assays either use nucleic acid-binding dyes such as ethidium bromide and SYBR Green I stain as reporter molecules (Higuchi, U.S. Pat. No. 5,994,056 and Wittwer et al., U.S. Pat. No. 6,174,670), or they use a minimum of two fluorophores immobilized on probes. The two fluorophores can either be donor-acceptor pairs individually attached to separate oligonucleotides (U.S. Pat. No. 6,174,670, and Di Cesare, U.S. Pat. No. 5,716,784), or they can be reporter-quencher pairs attached to a single oligonucleotide (Mayrand, U.S. Pat. No. 5,691,146, Pitner et al, U.S. Pat. No. 5,888,739 and Livak et al, U.S. Pat. No. 6,030787). Homogeneous assays using DNA binding dyes are convenient, but they provide limited sequence information. Methods based on two-dye systems can provide greater detection specificity, regardless of whether they are donor-acceptor or donor-quencher dye combinations, and are used in systems such as the Hybridization Probe assay (U.S. Pat. No. 6,174,670), the Taqman assay (U.S. Pat. No. 5,691,146), the Molecular Beacon assay (Tyagi et al, 1998. Nature Biotechnology 4:359-363) and its variant, the Scorpions primer system (Whitcombe et al, 1999. Nature Biotechnology 17:804-807).
In hybridization probe assays, two oligonucleotide probes are used to detect the presence of a particular sequence. Reporter signal is detected when fluorescence resonance energy transfer occurs between the donor dye on one probe and the acceptor on the other by bringing the two dyes into proximity through annealing of probes to target. Once the probes are hybridized, the area under one probe can be studied for possible sequence variances. This can be done by heating the sample and monitoring the temperature at which a loss in signal occurs by dissociation (or xe2x80x9cmeltingxe2x80x9d) of that probe. Sequence variances may be detected by a shift in the melting temperature (Tm) relative to a reference sample, and such Tm shifts can be predicted using software calculations (Schxc3xctz et al, 1999. BioTechniques 27:1218-1224). However, the area under the second probe may become a xe2x80x9cblind zonexe2x80x9d that is not analyzed for sequence variances. The presence of blind zones may be problematic when large segments of DNA need to be analyzed for sequence variances, and multiple probe pairs need to be employed.
The Taqman and molecular beacon assays both use a single oligonucleotide probe with both a reporter and a quencher dye attached. The oligonucleotide probe hybridizes to the target sequence, and the reporter and quencher are separated either by the exonuclease activity of the polymerase or due to change in conformation upon hybridization to the target sequence. Present methods result in relative difficulty in synthesizing these dual-labeled probes. Also, Taqman probes provide an indirect measure of hybridization, as signal continues to be generated once the reporter and quencher are separated by the exonuclease activity of the polymerase.
Changes in fluorescence efficiency of fluorophores by means other than energy transfer have been reported. Various dyes of the fluorescein family are sensitive to pH, and their emission intensities decrease at pHs lower than their pKa, and increase when the pH is close to or higher than the pKa (Sjxc3x6back et al, 1995. Spectrochim Acta A 51, L7). Also, fluorescein is quenched by more than 50% upon conjugation to biopolymers (Der-Balian et al, 1988. Analytical Biochemistry 173:9). These are general fluorescence changes that are induced by external factors. Also known is that the annealing of a fluorescent-labeled oligonucleotide and its unlabeled complementary strand may result in quenching of the probe fluorescence and a shift in the wavelength of emission upon the formation of duplex DNA (Cooper et al 1990. Biochemistry 29:9261-9268; Lee et al, 1994. Analytical Biochemistry 220:377-383; and Yguerabide et al, 1996. Analytical Biochemistry 241:238-247). Fluorescent intensity changes have also been shown using unbound dye and individual nucleotide or nucleoside molecules (Seidel et al, 1996 J. Phys Chem 100:5541-5553), RNA substrate-ribozyme interactions (Walter et al, 1997. RNA 3:392-404), and nucleic acid duplex formation using probes labeled with asymmetric cyanine dyes (Ishiguro et al 1996. Nucleic Acids Research 24:4992-4997; and Svanvik et al 2000. Analytical Biochemistry 281:26-35). However, these references do not teach the construction of probes that take advantage of sequence-dependent fluorescence.
Thus, the present invention is directed to oligonucleotide probes wherein each probe has a single fluorescent dye. The oligonucleotide probes are constructed such that hybridization of the probe to a target sequence affects the fluorescent emission of the fluorescent dye. In one embodiment of the invention, hybridization of the probe to the target sequence places the fluorescent dye in close proximity to a guanine residue, with resultant quenching of fluorescent emission. In another embodiment, the fluorescent entity replaces a base in the oligonucleotide probe structure, and upon hybridization this xe2x80x9cvirtual nucleotidexe2x80x9d is placed in a complementary position to a G residue, with resultant quenching of fluorescence. In other embodiments, probes are constructed such that hybridization results in an increase in fluorescent emission. In one such embodiment, the fluorescent entity is attached to a G residue, with increased fluorescence upon hybridization. In another such embodiment, the fluorescent entity is attached to a base analog, with resultant increase in fluorescence upon hybridization. In yet another embodiment of this invention, the fluorescent entity is attached to an internal residue via a flexible linker, with resultant change in fluorescent emission upon hybridization. Finally, various examples of probe systems are provided.
In one aspect of the invention a probe is provided for analyzing a target nucleic acid, the probe comprising a fluorescent detecting entity consisting essentially of an oligonucleotide having a sequence generally complementary to a locus of the target nucleic acid and a fluorescent label linked to a terminal nucleotide of the oligonucleotide, the oligonucleotide sequence of the probe being selected so that upon hybridization of the probe to the locus of the target nucleic acid the fluorescent label is positioned in proximity to a guanine residue of the target nucleic acid with resultant quenching of the fluorescent intensity of the fluorescent label. In one embodiment, the guanine residue is located at position 0, +1, +2, +3, or +4 relative to the position of the flourescent labeled terminal nucleotide.
In another aspect of this invention, a probe is provided for analyzing a target nucleic acid, the probe comprising an oligonucleotide having a sequence generally complementary to a locus of the target nucleic acid and further comprising a residue having a virtual nucleotide wherein a fluorescent dye is substituted for a base, and wherein the magnitude of flourescent emission from the fluorescent dye is altered by hybridization of the probe to the target nucleic acid.
In still another aspect of this invention, a fluorescence-based probe system is provided for analyzing a target nucleic acid, the probe system consisting essentially of a single-labeled polynucleotide comprising a sequence generally complementary to a locus of the nucleic acid and a fluorescent label attached thereto, whereby upon hybridization of the single-labeled polynucleotide to the locus of the nucleic acid the fluorescent label is positioned near a residue of the target nucleic acid with a resultant increase in fluorescent intensity of the fluorescent label. Various embodiments of these augmentation probes are provided.
In yet another aspect of this invention a probe for analyzing a target nucleic acid is provided, the probe comprising a fluorescent detecting entity consisting essentially of a single-labeled oligonucleotide having a sequence generally complementary to a locus of the target nucleic acid and a fluorescent label linked to an internal residue of the oligonucleotide, and wherein oligonucleotide sequence of the probe being selected so that upon hybridization of the probe to the locus of the target nucleic acid the magnitude of fluorescent emission from the fluorescent label is altered by hybridization of the probe to the target nucleic acid.
Additionally, an oligonucleotide probe is provided for detecting the presence of a target nucleic acid from the genus Salmonella the probe comprising a nucleotide sequence selected from the group consisting of
In another aspect of this invention methods are provided using the probes of this invention, in one such embodiment, a method is provided for determining the presence of a target nucleic acid sequence in a biological sample comprising combining a first single-labeled oligonucleotide probe with the sample, said first probe having an oligonucleotide sequence generally complementary to a locus of the target nucleic acid sequence and a fluorescent label linked to an end of the oligonucleotide sequence, the fluorescent label exhibiting an hybridization-dependent fluorescent emission, wherein hybridization of the first probe to the target nucleic acid sequence allows interaction of the fluorescent label with a guanine residue located on the target nucleic acid, thereby decreasing the magnitude of fluorescent emission from the label, illuminating the biological sample, and monitoring the hybridization-dependent fluorescent emission.
In a further aspect of this invention a method is provided for determining the presence of a target nucleic acid sequence in a biological sample comprising combining a single-labeled oligonucleotide probe with the sample, said probe having an oligonucleotide sequence generally complementary to a locus of the target nucleic acid sequence and a fluorescent label linked to a G residue of the oligonucleotide sequence, the fluorescent label exhibiting an hybridization-dependent fluorescent emission, wherein hybridization of the oligonucleotide probe to the target nucleic acid sequence alters interaction of the fluorescent label with the G residue, thereby increasing the fluorescent emission from the label, illuminating the biological sample, and monitoring the hybridization-dependent fluorescent emission.
In still a further aspect of this invention a method is provided for analyzing a sample comprising a target nucleic acid sequence, comprising the steps of combining the sample and an oligonucleotide probe to create a target-probe mixture, wherein the probe includes a virtual nucleotide having a fluorescent label positioned so that the magnitude of fluorescent emission from the fluorescent label is altered by hybridization of the probe to the target nucleic acid sequence, illuminating the mixture, and monitoring the fluorescent emission as a function of temperature.
In an additional aspect of this invention a method is provided for determining the presence a target nucleic acid sequence in a biological sample comprising combining the biological sample with a fluorescent detecting entity consisting essentially of a single-labeled oligonucleotide probe, wherein the single-labeled probe comprises an oligonucleotide having a sequence complementary to a locus of the target nucleic acid sequence, and having a fluorescent label exhibiting an hybridization-dependent emission attached thereto, wherein hybridization of the probe to a selected segment of the target nucleic acid sequence results in an increase in fluorescent emission of the fluorescent label, illuminating the biological sample, and monitoring the hybridization-dependent fluorescent emission. In one such embodiment the fluorescent label is linked to a base of the oligonucleotide probe and the base is selected from the group consisting of 5-nitroindole, 4-nitroindole, 6-nitroindole, and 3-nitropyrrole deoxynucleosides. In another such embodiment, the fluorescent label is attached to a guanine residue and the monitoring step includes monitoring the increased fluorescent emission from the fluorescent label upon hybridization of the probe to the target nucleic acid. In yet another embodiment, the fluorescent label is selected from the group consisting of cyanine dyes and LCRed 705.
An additional aspect of this invention is a kit for analyzing a biological sample comprising a nucleic acid sequence, comprising a fluorescent detecting entity consisting essentially of a single-labeled oligonucleotide probe having an oligonucleotide linked to a fluorescent label, wherein said probe is configured to hybridize to a locus of the segment so that the magnitude of fluorescent emission from the fluorescent label is increased by hybridization of the probe to the locus; and components for amplification of the nucleic acid sequence.
Additional features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.